A5, a mutant BHK cell line resistant to Methotrexate, overproduces DHFR by 140-fold as compared to the parental B1 line. The rate of DHFR synthesis is approximately 140-fold greater in A5 cells than 1 cells, while all other physical parameters of the enzymes are identical in both lines, including the rates of degradation. This and other indirect evidence strongly suggests that A5 cells synthesize considerably more DHFR mRNA than B1 cells due to a cis regulatory mutation. We propose to clone the DHFR operon (the structural gene and adjacent cis regulatory sites) to permit the study of transcriptional regulatory processes in vitro with the ultimate goal being a clearer understanding of these processes on the molecular level. We plan to approach this goal by studying the A5 and B1 operons to determine what regulatory element is defective in the A5 cells. We propose three procedures for cloning the DHFR operon to provide the DNA template coding for the structural gene and the cDNA to it. Through hybridization experiments with this DNA, we will confirm that A5 cells overproduce DHFR mRNA and determine the gene reiteration frequency in both the A5 and B1 lines. Using the cloned DHFR operon DNA, we will develop an in vitro transcription system that faithfully reproduces the observed in vivo quantitative difference in transcription from the A5 and B1 templates. This system will then allow us to examine the regulatory mechanisms controlling expression of the DHFR structural gene.